PROJECT SUMMARY/ABSTRACT Cancer remains a leading cause of mortality within the US, responsible for 1 in 4 deaths. Immunotherapies have ushered in a new age of cancer treatment, leveraging the potency of the immune system to restrict cancer without many of the side effects of conventional therapies. These immunotherapies have primarily targeted immune responses specific for tumor antigens (tAg). Although tAg-specific T cell immunotherapies have proven powerful tools in combatting cancer, their success is context-dependent, displaying limited efficacy in most solid tumors. This is largely owed to chronic T cell receptor (TCR) binding to cognate tAg, leading to permanent cellular dysfunction. Solid tumors comprise the majority of cancer cases and deaths, thus, it is essential to exploit other cellular modalities in immunotherapies. Recently, it has become apparent that tAg-nonspecific ?bystander? T cells are observed and often outnumber tAg-specific T cells in solid tumors. Although their function within tumors is unknown, bystander T cells can exert cytotoxic effector function once activated by inflammation in a number of contexts. My unique approach leveraging T cells with defined T cell receptors (TCRs) allows me to determine the mechanisms that dictate bystander T cell entry into the tumor and if they maintain the ability to respond to stimulation once tumor-resident. To appreciate the heterogeneity of tumor microenvironments, I will employ multiple animal models of solid tumors. My objectives are two-fold: First, I want to test how bystander T cells migrate to the tumor and if they are spared from dysregulation due to their inability to recognize tAg. Second, I want to test if the dichotomous effects of bystander T cells can be therapeutically leveraged to improve anti- tumor responses. At homeostasis, bystander T cells can simply deny other immune cells access to targets, hindering antigen (Ag)-specific immune responses. Once activated by inflammation, bystander T cells rapidly acquire effector function and directly kill target cells in an innate-like manner. To achieve these objectives, I will employ my expertise in 28-color flow cytometry to interrogate cell phenotype, activation, and functional capacity. I will complement my use of flow cytometry with 3-dimensional immunofluorescence, which will uncover sub- anatomic immune cell organization within the tumor. Hypothesis: My central hypothesis is that bystander T cells in solid tumors remain functional and can be therapeutically leveraged in cancer specifically. I will test this hypothesis through two independent aims: AIM 1: Test the hypothesis that bystander T cells are recruited into tumors by CXCR3 and remain functional in the tumor microenvironment. AIM 2: Test the hypothesis that anti- tumor immune responses can be enhanced by activated bystander T cells or targeted depletion of tissue-resident bystanders. My proposed experiments will elucidate the role of bystander T cells in tumor both with and without interventions, with the goal of developing interventions to improve cancer outcomes, which is in alignment with the mission of the NCI and NIH.